Droplet microfluidics is a relative new but rapidly advancing field. It provides methods to manipulate liquid droplets and/or the particles in the droplets by employing mechanisms such as electrowetting [WO2008147568, Electrowetting Based Digital Microfluidics], electrophoresis [WO2014036914, Method and Device for Controlling, Based on Electrophoresis, Charged Particles in Liquid], and dielectrophoresis [WO2014036915, Dielectrophoresis Based Apparatuses and Methods for the Manipulation of Particles in Liquids], etc. It provides droplet operation capabilities such as droplet dispensing and transport, merging and mixing of multiple droplets, splitting one droplet to two (or more) daughter droplets, incubation, waste disposal, particles (such as DNA/RNA/protein molecules, cells, beads, etc.) redistribution/enrichment/separation, etc. Droplet microfluidics provides the capability to handle all the basic steps of liquid analysis, including sampling, sample preparation, reaction, detection, and waste handling, etc. It can practically handle droplets with volume ranging from a few pico-liters to tens of microliters—a span of more than 6 orders of magnitude. It finds applications in medical diagnostics, cancer screening, drug discovery, food safety inspection, environmental monitoring, forensic analyses, and many others. Besides miniaturization and integration, it offers other advantages such as low cost, automation, parallelism, high throughput, low energy consumption, etc.
A typical digital microfluidic (DMF) device consists of two solid substrates separated by a spacer to form a gap in-between. Liquids are operated in the gap in a discrete fashion, i.e., in the format of droplets. Different from channel based microfluidics, in digital microfluidics, the liquid/droplet path can be changed during run-time by the control software, and the droplets can be operated individually. Digital microfluidics truly fulfill the promise of the lab-on-a-chip concept, which is to handle all the basic steps of an analysis, including sampling, sample preparation, reaction, detection, and waste handling, etc. Digital microfluidics shares great similarities with bench based liquid handling. Established bench based protocols can be easily adapted to the digital microfluidics format.
Chemical and biochemical reactions often need a well-regulated temperature profile to perform efficiently. For example, in DNA amplification methods like Polymerase Chain Reaction (PCR), Ligase Chain Reaction (LCR), transcription-based amplification, and restriction amplifications, etc., the reactions require cycling between higher denaturing temperatures and lower polymerization temperatures. Other nucleic acid amplification methods require the reaction to take place at a specified constant temperature, such as Self-Sustained Sequence replication (3SR), Rolling-Circuit Amplification (RCA), Strand Displacement Amplification (SDA), and Loop Mediated Amplification (LAMP), Helicase-Dependent Amplification (HAD), etc. The detection of the fluorescence intensity changes when a DNA molecule going through a well-defined temperature profile can provide great insight such as the presence and identity of single nucleotide polymorphisms (SNP)—a process called melting curve analysis.
Well-regulated temperature control is also important in the processing of RNA and protein molecules, for example, real time RT-PCR (Reverse Transcription-Polymerase Chain Reaction and Isothermal RNA Signal Generation (IRSG) for RNA detection and real time immuno-PCR and IAR (Isothermal Antibody Recognition) for protein detections. Cell lysis is generally temperature dependent too.
Due to its great sensitivity, PCR is one of the most commonly used nucleic acid amplification and quantification methods in clinical diagnostics, forensic science, and environmental science, etc. While the reaction at molecular level is typical very fast, the speed of PCR is often limited by the time it takes to cycle through different needed temperatures. Fast/ultrafast PCRs are often highly desirable, especially in the situation of infectious disease diagnostics, bio-warfare and pathogen identification, forensic analyses, etc. It is even more desirable to achieve fast/ultrafast PCRs with low power consumption, compact size and simple operation.
Microfluidic thermal management has long been a major issue. Many techniques have been explored to regulated the temperature within microfluidic systems. They range from the use of Peltier [Maltezos, G. et al, Microfluidic polymerase chain reaction, Appl. Phys. Lett. 2008, 93, 243901:1-243901:3], Joule heating [Mavraki, E. et al, A continuous flow μPCR device with integrated micro-heaters on a flexible polyimide substrate. Procedia Eng. 2011, 25, 1245-1248], endothermal chemical reactions [Guijt, R. M. et al, Chemical and physical processes for integrated temperature control in microfluidic devices. Lab Chip 2003, 3, 1-4], microwaves [Shaw, K. J. et al, Rapid PCR amplification using a microfluidic device with integrated microwave heating and air impingement cooling. Lab Chip 2010, 10, 1725-1728], and Lasers [Kim, H. et al, Laser-heated reactions in nanoliter droplet arrays. Lab Chip 2009, 9, 1230-1235], etc. Now, people are still looking for reliable, easy-to-use, and economical ways to regulate temperature in a microfluidic system.
Patent WO2009003184 [Digital Microfluidics Based Apparatus for Heat-Exchanging Chemical Processes] presented a device design and method to use external temperature control modules to create different temperature zones on the device. Heat-exchanging chemical processes such as PCR can be performed by transporting the reaction droplets back and forth between different temperature zones.
However, the above mentioned use of external temperature control modules on a microfluidic device has its limitations. When contact temperature control modules are used, the bottom and cover substrates of the microfluidic device act as diffusers, which limits the temperature resolution at the device gap where reactions take place. When using non-contact heating methods such as photonic-based heating [Ultrafast photonic PCR, J H Son, et al., Light: Science & Applications (2015) 4] or microwave heating [K J Shaw, et al., Rapid PCR amplification using a microfluidic device with integrated microwave heating and air impingement cooling. Lab Chip 10:1725], complex and expensive focusing mechanisms would be needed to achieve high spatial temperature resolution.
In one embodiment, provided are simple and cost effective approaches to implement high spatial resolution temperature control in a droplet microfluidic device. By disposing the heating electrodes on the cover plate surface facing the device gap in which droplets are manipulated, many different temperature zones can be created in the device gap to provide ideal reaction environments for different chemical/biochemical reactions. A shielding electrode, which is typically grounded electrically, is disposed to cover (at least partially) the heating electrodes. This shielding electrode prevents the droplets from being affected by the possible electric and/or magnetic field(s) generated by the heating electrodes. External temperature control modules, such as Peltiers or water/air cooling blocks, can be used together with the heating electrodes increase the temperature control range, for example, to below the room temperature.
The temperature of a controlled region in the gap of a droplet microfluidic device can range from −20° C. (minus 4° C.) to 200° C., and preferably from 0° C. to 120° C., and more preferably from 20° C. to 98° C.
In one aspect, the heating electrodes can be integrated with feedback control. For example, a typical implementation of a heating electrode is to deposit a layer of conductive material at specific thickness, width and length, so that it has a specific resistance. When an electric current is going through the heating electrode, the heat is generated—Joule heating. The heating electrode is can be called a resistive heater. In general, the resistance value of a resistive heater is temperature dependent. By measuring the resistance change, the temperature change (compared to a starting point) can be calculated. This means the resistive heater can also be used as temperature sensor. Other temperature sensors such as, but not limiting to, thermal couple, thermistor and separated resistance temperature detector (RTD) can be used to continuously monitor the temperature profile of the device. These sensors can be placed in the device gap, or on the top or bottom plate(s) of the device temporarily for temperature calibration or permanently to enable closed-loop temperature control during run-time.
Melting curve analysis is an assessment of the dissociation-characteristics of double-stranded DNA during heating. The information gathered can be used to infer the presence of and identity of single nucleotide polymorphisms. The present invention provides methods for implementing temperature sweeps needed for melting curve analyses. In one aspect, the invention provides methods to implement temperature changes through spatial variation. Thus, two or more regions of the device can be set to different temperatures (proper for melting curve analysis), at thermal equilibrium, a path (or multiple paths) of continuous temperature change from the temperature at the highest temperature region to the temperature at the lowest temperature region can be designed on the device. A droplet of PCR product can be moved along this path (or paths), and the fluorescence measured as the PCR product moves along the path. The change in fluorescence can be used to obtain the melting curve for the DNA strand. In another aspect of the invention, the droplet of PCR product can be made to remain stationary at one location and the temperature(s) at the location can be changed. The fluorescence data can be collected at said location to obtain the melting curve for the DNA strand.
DNA sequencing is the process of determining the precise order of the four chemical building blocks, called “bases,” that make up the DNA molecule. Sequence data, among many things, can highlight changes in a gene that may cause disease. Although DNA sequencing technology and platforms being rapidly developed, the sample processing, also called library preparation, lacks behind. This is an area that droplet microfluidics can offer help.
Library preparation typically has a few major steps—genomic DNA fragmentation, end repair, adding ‘A’ bases to the 3′ end of the DNA fragments, adapter ligation to DNA fragments, ligation products purification, PCR amplification of the adapter-modified DNA fragments, etc. Different steps often require different temperature profile. This invention provides a convenient approach to create the needed temperature profile on the droplet microfluidic device to enable fast library preparation.